Human CNTF was discovered by Silvio Varon et al. in 1979 to be a factor for promoting the survival of ciliary ganglions which are parasympathetic nerves (Brain Res., 173, 29-45 (1979)). Subsequently, the purification and cloning of human CNTF were reported in G. Barbin et al., J. Neurochem., 43, 1468-1478 (1984); L. F. Lin et al., Science, 246, 1023 (1989); P. Masiakowski et al., J. Neurochem., 57, 1003 (1991); A. Negro et al., Dur. J. Biochem, 201, 289 (1991); J. R. McDonald et al., Biochim. Biophys. Acta, 70, 1090 (1991); WO 90/7341 and WO 91/4316.
In connection with the pharmacological activities of human CNTF in vitro, there are the following reports on the survival promoting activity of human CNTF on hippocampal and septal GABA-mediated neurons (N. Y. IP et al., N. Neursci., 11, 3124-3134 (1991)); on optic neurons (H. D. Hoffman, J. Neurochem., 51, 109-113 (1988)); on sensory neurons (G. Barbin et al., J. Neurochem., 43, 1468-1478 (1984)); on parasympathetic neurons (Y. Arakawa et al., J. Neursci., 10, 3507-3515 (1990)) and on motor neurons (M. Sendtner et al. J. Cell Sciences supple., 15, 103-109 (1991)); and on the differentiation activity of human CNTF into cholinergic neurons (S. Saadat et al., J. Cell Biol., 108, 1807-1816 (1989) and U. Ernsberger et al., Neuron, 2, 1275-1284 (1989)) and into type 2A astrocytes (D. J. Anderson et al., TINS, 12, 83 (1989)); the in vivo activities are reported on the survival promoting effect on septal cholinergic neurons in a fimbria-fornix trunsection model (T. Hagg et al., Neuron, 8, 145 (1992))1D on the survival promoting activity of human CNTF on motor neurons in an axotomy model (M. Sendtner et al., Nature, 345, 440-441 (1990)); on the survival promoting activity of human CNTF on motor neurons in mice with genetic motor disorders (M. Sendtner et al. in Nature, 358, 502-503 (1992)); on the protecting effect of human CNTF for substantia nigra dopaminergic neurons in a substantia nigra-striatun trunsection model (T. Hogg & S. Varon, Proc. Natl. Acad. Sci., USA, 90, 6315-6319 (1993)); on the protecting effect for a photoreceptor (M. M. LaVail et al., Proc. Natl. Acad. Sci., USA, 89, 11249-11253 (1992)); on the protecting effect for optic neurons (K. Unoki & M. M. LaVail in Investigative Ophthalmology & Visual Sciences 35, 907-915 (1994)); and so on.
As reported above human CNTF acts on neurons to exert the activities of promoting the survival of neurons, accelerating the neurite outgrowth of neurons and stimulating the synthesis of neurotransmitters. Human CNTF is thus expected to be useful for the treatment of trauma-induced nervous disorders, diseases caused by atrophy or denaturation of neurons including Alzheimer's disease, cerebrovascular dementia and amyotrophic lateral sclerosis (J. E. Springer, DN & P, 4, 394 (1991) R. M. Lindsay, Neurobiol. of Aging, 15, 249-251 (1994) and R. M. Lindsay, TINS, 17, 182 (1994)).
It is also reported that human CNTF effectively displays, in combination with brain-derived neurotrophic factor (BDNF) a protecting activity on nervous disorders in a neurodegenerative animal model (Wobbler mice) (H. Mitsumoto et al., Science, 265, 1107-1110 (1994)). It is suggested that human CNTF will be an effective drug for the treatment of nervous disorders, not only by human CNTF alone but also in combination with other neurotrophic factors (R. Nishi, Sciences, 265, 1052-1053 (1994)).
However, in clinical trials of wild type human CNTF currently used in USA for the treatment of amyotrophic lateral sclerosis, a considerably large doses e.g., 6 mg/week, of human CNTF is administered (Bio World Today, Sep. 8, 1993). Generally, in consecutive administration of a proteinaceous preparations an increased dose tends to involve such problems that side effects might be caused due to the generation of an autoantibody and that the production costs might increase. In fact, the formation of antibodies is observed during the CNTF evaluation in phase II and phase III in USA (BIO World Todays Sep. 8, 1993). Furthermore, an additional problem arises to cause other side effects (BIO World Todays Jun. 24, 1994 and Science, 264., 772-774 (1994)). It is therefore expected that the foregoing problems caused by the use of wild type human CNTF will be improved, if a mutant of human CNTF having a higher specific activity than that of the wild type is employed.
It is reported that human CNTF is composed of 200 amino acid residues, abundant in .alpha.-helix and contains 53% .alpha.-helix and 9% .beta.-turn structure (A. Negro et al., J. Neurosci. Res., 29, 251 (1991)). Analysis of the secondary structure suggests that human CNTF would take a structure similar to that of .alpha.-helical cytokines. It is reported that human CNTF has a 4-helix bundle structure which is commonly observed in all .alpha.-helical cytokines, such as growth hormone (hereinafter abbreviated as GH), prolactin (hereinafter abbreviated as PRL), erythropoietin (hereinafter abbreviated as EPO), granulocyte colony-stimulating factor (hereinafter abbreviated as G-CSF) oncostatin H (OSM), leukemia inhibitory factor (hereinafter abbreviated as LIF) and some interleukins (J. F. Bazan, Neuron, 7, 197 (1991)). The four helices are designated successively as A, B, C and D from the N terminus.
Proteins having a 4-helix bundle structure are classified into a long-chain group comprising 160 to 200 amino acid residues in length and a short-chain group comprising 105 to 145 amino acid residues in length (J. L. Boulay & W. E. Paul, Curr. Biol., 3, 573 (1993) and S. Sprang & J. Bazan, Curr. Opin. Struct. Biol., 3, 815 (1993)). The long-chain group includes GH, PRL, EPO, G-CSF, LIF, interleukin 6 (hereinafter abbreviated as IL-6) and the like; the short-chain group includes interleukin 2 (hereinafter abbreviated as IL-2), interleukin 4 (hereinafter abbreviated as IL-4) and granulocyte macrophage colony-stimulating factor (hereinafter abbreviated as GM-CSF) and the like. Human CNTF is one of the long-chain group proteins and predicted to be more closely akin to the proteins of this group.
With respect to the proteins mentioned above, many results on the structure-activity relationship have been reported to date. These reports point out that the amino acid residues between helices A and B (hereinafter referred to as AB loop region) and helix D region are important for expressing the biological activity of many .alpha.-helical cytokines. Similar reports are also seen on IL-6 (R. Savino et al., Proc. Natl. Acad. Sci. USA, 90, 4067 (1993) C. Lutticken et al., FEBS Lett. 282, 265 (1991), J. P. J. Brakenhoff et al., J. Immunol., 145, 561 (1990) and X. Li et al., J. B. C, 268, 22377 (1993)). Regarding GH, a similar importance is observed with GH mutant constructed (B. C. Cunningham et al., Science, 247, 1461 (1990)) and the structure of the GH mutant has been directly determined by crystallography of the complex of GH and a GH receptor (A. M. DeVos et al., Science, 255, 306 (1992)).
On the other hand, Bazan reported that there is a similarity of amino acid sequence, which is called D1 motif, in the boundary region (hereinafter referred to as D1 cap region) between the amino acid residues which link helix C and helix D (hereinafter referred to as CD loop region) and helix D (J. F. Bazan, Neuron, 7 197 (1991)). The amino acid sequence of D1 motif is represented by: EQU -.phi.-(F/W)-(E/Q)-(K/R)-(K/R)-.phi.-X-G-
wherein .phi. is a hydrophobic residue and X is any residue. The D1 motif corresponds to the amino acid residues 151-158 in human CNTF. This region is considered as corresponding to the respective amino acids of, e g., LIF (amino acid residues 154-161) OSM (amino acid residues 159-166) IL-6 (amino acid residues 157-164) and IL-11 (amino acid residues 148-155). The boundary region of CD loop and helix D was reported to be important for receptor binding of IL-6 (J. P. J. Brakenhoff et al., J. B. C., 269, 86 (1994)) and LIF (R. C. Robinson et al., Cell, 77, 1101 (1994)). However, the amino acid sequence in the D1 cap region of IL-6 lacks similarity with the D1 motif and hence it has not been identified what amino acids on the D1 motif are important. In the same regions LIF has great similarity in the amino acid sequence to the D1 motif but what amino acid residues are important has not been identified. There is no other report on other .alpha.-helical cytokines to suggest that the foregoing region would be important for their biological activity.
In human CNTF, three receptors, i.e., CNTF receptor .alpha. (hereinafter abbreviated as CNTF-R.alpha.), LIF receptor (hereinafter abbreviated as LIF-R) and gp130 are responsible for the signal transduction into cells. gP130 is a constituent of the receptor not only for human CNTF but for IL-6, LIF, OSM and interleukin 11 (hereinafter abbreviated as IL-11) in common (S. Davis et al., Science, 253, 59 (1991), S. Davis et al, ibid., 260, 1805 (1993), D. P. Gearing et al., ibid., 255, 1434 (1992) and N. Y. Ip et al., Cell, 69, 1121 (1992)). In view of the aforesaid structural similarity and also the same receptor being commonly involved, it is highly likely that in human CNTF, IL-6, LIF, OSM and IL-11 the amino acid residue corresponding to the same region in each ligand will be associated with receptor binding
Other .alpha.-helical cytokines which have structural similarity to CNTF have also been studied for clinical application to develop for the treatment of various diseases With respect to many cytokines including GM-CSF, macrophage colony stimulating factor (hereinafter abbreviated as H-CSF), interleukin 3 (hereinafter abbreviated as IL-3) and IL-6, clinical tests have been performed or are under consideration, as described in ZOKETSU INSHI (Hematopoietic Factor), 5, 6-86 (1994).
There is a possibility that, even in the case of other .alpha.-helical cytokines, the use of mutant proteins having higher activities than wild type proteins may effectively improve the problems similar to those of wild type CNTF.